This study represents a major new direction in my laboratory to investigate the regulatory mechanisms involved in the transport of glucose in mammalian brain. Two glucose transporter isoforms, GLUT1 and GLUT3, have been identified in brain. In whole brain GLUT1 is detected as two molecular weight forms: a 55kDa form which is concentrated in the endothelial cells of the blood-brain barrier and a 45 kDa form present in vascular-free cortical membranes, as well as in primary cultures of neurons and glia. In the rat, GLUT3 is expressed exclusively in the brain but is notably absent from the blood-brain barrier of both rat and human. Ontogeny studies indicate that in the rat brain, the concentration of GLUT3 and both forms of GLUT1 progressively increase from the fetus through 30 days postnatally. We have detected GLUT3 in adult rat brain in all regions except the adenohypophysis and pineal gland, in primary neuronal but not astrocytic cultures, and in cultured cells of neuronal origin, e.g. PC12 and NG108-15 cells. Studies with primary cultures revealed an increase in the GLUT 1 and 3 mRNA and protein levels which peaked at 4 and 6 days respectively, coincident with maximal glucose transport capacity and complete cellular differentiation. Quantitative studies using either a specific [3H]-bis-mannose photolabel or [35S] methionine incorporation indicated that the concentration of GLUT3 is five times greater than GLUT1 in these cultured neurons. In serum-free cultures GLUT1 and GLUT3 expression appears to be unaffected by ambient glucose concentrations but is reduced by lowering the potassium concentrations.